Challenges in mass spectrometry-based quantification of bioactive peptides: A case study exploring the neuropeptide Y family

The study of biologically active peptides is critical to the understanding of physiological pathways, especially those involved in the development of disease. Historically, the measurement of biologically active endogenous peptides has been undertaken by radioimmunoassay, a highly sensitive and robust technique that permits the detection of physiological concentrations in different biofluid and tissue extracts. Over recent years, a range of mass spectrometric approaches have been applied to peptide quantification with limited degrees of success. Neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP) belong to the NPY family exhibiting regulatory effects on appetite and feeding behavior. The physiological significance of these peptides depends on their molecular forms and in vivo concentrations systemically and at local sites within tissues. In this report, we describe an approach for quantification of individual peptides within mixtures using high-performance liquid chromatography electrospray ionization tandem mass spectrometry analysis of the NPY family peptides. Aspects of quantification including sample preparation, the use of matrix-matched calibration curves, and internal standards will be discussed. This method for the simultaneous determination of NPY, PYY, and PP was accurate and reproducible but lacks the sensitivity required for measurement of their endogenous concentration in plasma. The advantages of mass spectrometric quantification will be discussed alongside the current obstacles and challenges. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 98: 357–366, 2012.

Parental bonding, family systems, and environmental predictors of adolescent homelessness

A multicausal model of adolescent homelessness is proposed, based upon the notion that homeless youth suffer from emotional, social, and cultural deprivation. The model was tested in a sample of homeless adolescents (n = 54) and a similar, but not homeless, control group (n = 58). Emotional deprivation was assessed on the Parental Bonding Inventory (Parker, Tupling,&Brown, 1979), whereas social and cultural deprivation were assessed on the Family Environment Scale (Moos&Moos, 1981). The homeless adolescents were found to be significantly more deprived emotionally, socially, and culturally than the controls. The results indicate support for a deprivation model of adolescent homelessness with implications for public policy and intervention planning.

“I would have lost the respect of my friends and family if they knew I had bent the road rules” : parents, peers, and the perilous behaviour of young drivers

Young novice drivers are at considerable risk of injury on the road. Their behaviour appears vulnerable to the social influence of their parents and friends. The nature and mechanisms of parent and peer influence on young novice driver (16–25 years) behaviour was explored via small group interviews (n = 21) and two surveys (n1 = 1170, n2 = 390) to inform more effective young driver countermeasures. Parental and peer influence occurred in preLicence, Learner, and Provisional (intermediate) periods. Pre-Licence and unsupervised Learner drivers reported their parents were less likely to punish risky driving (e.g., speeding). These drivers were more likely to imitate their parents and reported their parents were also risky drivers. Young novice drivers who experienced or expected social punishments from peers, including ‘being told off’ for risky driving, reported less riskiness. Conversely drivers who experienced or expected social rewards such as being ‘cheered on’ by friends – who were also more risky drivers – reported more risky driving including crashes and offences. Interventions enhancing positive influence and curtailing negative influence may improve road safety outcomes not only for young novice drivers, but for all persons who share the road with them. Parent-specific interventions warrant further development and evaluation including: modelling safe driving behaviour by parents; active monitoring of driving during novice licensure; and sharing the family vehicle during the intermediate phase. Peer-targeted interventions including modelling of safe driving behaviour and attitudes; minimisation of social reinforcement and promotion of social sanctions for risky driving also need further development and evaluation.

Proteomic identification of putative microRNA394 target genes in Arabidopsis thaliana identifies MLP family members critical for normal development

Expression of the F-Box protein Leaf Curling Responsiveness (LCR) is regulated by microRNA, miR394, and alterations to this interplay in Arabidopsis thaliana produce defects in leaf polarity and shoot apical meristem (SAM) organisation. Although the miR394-LCR node has been documented in Arabidopsis, the identification of proteins targeted by LCR F-box itself has proven problematic. Here, a proteomic analysis of shoot apices from plants with altered LCR levels identified a member of the Major Latex Protein (MLP) family gene as a potential LCR F-box target. Bioinformatic and molecular analyses also suggested that other MLP family members are likely to be targets for this post-translational regulation. Direct interaction between LCR F-Box and MLP423 was validated. Additional MLP members had reduction in protein accumulation, in varying degrees, mediated by LCR F-Box. Transgenic Arabidopsis lines, in which MLP28 expression was reduced through an artificial miRNA technology, displayed severe developmental defects, including changes in leaf patterning and morphology, shoot apex defects, and eventual premature death. These phenotypic characteristics resemble those of Arabidopsis plants modified to over-express LCR. Taken together, the results demonstrate that MLPs are driven to degradation by LCR, and indicate that MLP gene family is target of miR394-LCR regulatory node, representing potential targets for directly post-translational regulation mediated by LCR F-Box. In addition, MLP28 family member is associated with the LCR regulation that is critical for normal Arabidopsis development.

The Expanded Human Kallikrein (KLK) gene family : genomic organisation, tissue specific expression and potential functions

The tissue kallikreins are serine proteases encoded by highly conserved multigene families. The rodent kallikrein (KLK) families are particularly large, consisting of 13 26 genes clustered in one chromosomal locus. It has been recently recognised that the human KLK gene family is of a similar size (15 genes) with the identification of another 12 related genes (KLK4-KLK15) within and adjacent to the original human KLK locus (KLK1-3) on chromosome 19q13.4. The structural organisation and size of these new genes is similar to that of other KLK genes except for additional exons encoding 5 or 3 untranslated regions. Moreover, many of these genes have multiple mRNA transcripts, a trait not observed with rodent genes. Unlike all other kallikreins, the KLK4-KLK15 encoded proteases are less related (25–44%) and do not contain a conventional kallikrein loop. Clusters of genes exhibit high prostatic (KLK2-4, KLK15) or pancreatic (KLK6-13) expression, suggesting evolutionary conservation of elements conferring tissue specificity. These genes are also expressed, to varying degrees, in a wider range of tissues suggesting a functional involvement of these newer human kallikrein proteases in a diverse range of physiological processes.

Work/Family Balance: HRM Policy and Practice in Australia

An employee's inability to balance work and family responsibilities has resulted in an increase in stress related illnesses. Historically, research into the nexus between work and family has primarily focused on the work/family conflict relationship, predominately investigating the impact of this conflict on parents, usually mothers. To date research has not sufficiently examined the human resource management practices that enable all parents to achieve a balance between their work and family lives. This paper explores the relationship between contemporary family friendly HRM policies and employed parents perceptions of work/family enhancement, work/family satisfaction, propensity to turnover, and work/family conflict. Self-report questionnaire data from 326 men and women is analysed and discussed to enable organisations to consider the use of family friendly policies and thus create a convergence between the well-being of employees and the effectiveness of the organisation.